Assessment and significance of protein–protein interactions during development of protein biopharmaceuticals

Abstract

Early development of protein biotherapeutics using recombinant DNA technology involved progress in the areas of cloning, screening, expression and recovery/purification. As the biotechnology industry matured, resulting in marketed products, a greater emphasis was placed on development of formulations and delivery systems requiring a better understanding of the chemical and physical properties of newly developed protein drugs. Biophysical techniques such as analytical ultracentrifugation, dynamic and static light scattering, and circular dichroism were used to study protein-protein interactions during various stages of development of protein therapeutics. These studies included investigation of protein self-association in many of the early development projects including analysis of highly glycosylated proteins expressed in mammalian CHO cell cultures. Assessment of protein-protein interactions during development of an IgG1 monoclonal antibody that binds to IgE were important in understanding the pharmacokinetics and dosing for this important biotherapeutic used to treat severe allergic IgE-mediated asthma. These studies were extended to the investigation of monoclonal antibodyantigen interactions in human serum using the fluorescent detection system of the analytical ultracentrifuge. Analysis by sedimentation velocity analytical ultracentrifugation was also used to investigate competitive binding to monoclonal antibody targets. Recent development of high concentration protein formulations for subcutaneous administration of therapeutics posed challenges, which resulted in the use of dynamic and static light scattering, and preparative analytical ultracentrifugation to understand the self-association and rheological properties of concentrated monoclonal antibody solutions.

Global multi-method analysis of interaction parameters for reversibly self-associating macromolecules at high concentrations

“…At this point k Dθ and k S θ are of equal magnitude but opposite sign, here ≈3.5 mL/g. Based on theory of hydrodynamic interactions on diffusion coefficients by Anderson and Reed 52 , which does not consider volume exclusion contributions 48 , it has been argued that under these conditions negative k D -values signify attractive interactions only if they are less than − 5.3 mL/g 34 , 53 . However, considering the fact that B 2 is also less than the obligatory excluded volume repulsion, we would argue that compensating attractive interactions must obviously be at play.…”

Targeted IgMs agonize ocular targets with extended vitreal exposure

“…We evaluated the viscosity of our IgM hexamer versus historical data surveying a range of IgG viscosities ( Figure 2 ). 38 Viscometer readings for IgMs show the expected log-linear response. Under viscosities conducive to intravitreal administration without patient pain or undue backpressure on the syringe (~10-50 cP), the median reported IgGs were limited to ~120-150 mg/ml, while the IgM was limited to 100–130 mg/ml.…”

Insights into the Conformation and Self-Association of a Concentrated Monoclonal Antibody using Isothermal Chemical Denaturation and Nuclear Magnetic Resonance

“…15,18,39 Furthermore, the increase in negative valence with increasing pH and the decrease of mutual distance with increasing concentration, promotes attractive interactions between oppositely charged surface areas of mAb1 that may lead to transient networks and clustering. 17,40…”